Active immunization using a siderophore receptor protein

ABSTRACT

The invention provides a vaccine for immunizing poultry and other animals against infection by a gram-negative bacteria, and a method of immunizing an animal using the vaccine. The vaccine may contain purified siderophore receptor proteins derived from a single strain or species of gram-negative bacteria or other organism, which are cross-reactive with siderophores produced by two or more strains, species or genera of gram-negative bacteria. The invention further provides a process for isolating and purifying the siderophore receptor proteins, and for preparing a vaccine containing the proteins. Also provided is a method for diagnosing gram-negative sepsis.

BACKGROUND OF INVENTION

The economic impact of infectious diseases in the poultry industry iswell-appreciated. Immunization of birds has helped reduce the cost ofproduction by decreasing the incidence of gastrointestinal, respiratoryand systemic diseases. While vaccines provide adequate immunity forthose pathogens against which a flock has been immunized, there are fewvaccines which can provide broad-based cross-protection againstunanticipated diseases or against those diseases for which an animal hasnot been specifically vaccinated.

A number of important diseases of domestic poultry are caused bybacteria able to invade host tissues, such as Salmonella spp.,Escherichia spp. and Pasteurella spp. While many vaccines are availablefor immunization against individual species and serotypes, none providecross-protection or stimulate broad-based immunity against multipleserotypes, species or genera.

One essential factor required for a bacteria to induce clinical diseaseis the ability to proliferate successfully in a host tissue. Iron is anessential nutrient for the growth of gram-negative bacteria in vivo, butis virtually unavailable in mammalian and/or avian tissues because theiron is either intracellular or extracellular, complexed with highaffinity, iron-binding proteins, for example, transferring in blood andlymph fluids and lactoferrin in external secretions. In normal tissues,the concentration of iron is approximately 10⁻¹⁸M, far below thatrequired for bacterial growth.

To circumvent these restrictive conditions, pathogenic bacteria haveevolved high affinity iron transport systems produced under low ironconditions, which consist of specific ferric iron chelaters,“siderophores,” and iron-regulated outer membrane proteins (TROMPs)and/or siderophore receptor proteins (SRPs) which are receptors forsiderophores on the outer membrane of the bacterial cell. Siderophoresare synthesized by and secreted from the cells of gram-negative bacteriaunder conditions of low iron. Siderophores are low molecular weightproteins ranging in molecular mass from about 500 to about 1000 MW,which chelate ferric iron and then bind to IROMPs in the outer bacterialmembrane which, in turn, transport the iron into the bacterial cell.Although the use of IROMPs as immunogens has been considered, theseproteins have not been examined for such use, at least in part, due toan inability to extract these proteins from bacterial membranes in highvolume and with a desired level of purity and immunogenic quality.

Accordingly, an object of the invention is to provide a method forobtaining high amounts of immunogenic quality siderophore receptorproteins from Escherichia coli, Salmonella, Pasteurella, and othergram-negative bacteria. Another object is to provide a vaccine forimmunizing poultry and other animals against these bacteria. Yet anotherobject is to provide a vaccine for cross-protection against multipleserotypes, species and/or genera of bacteria belonging to the familyEnterobacteriaceae and/or Pasteurellaceae. A further object is toprovide a diagnostic assay to monitor and/or profile sepsis andsubclinical disease caused by gram-negative bacteria under fieldconditions.

SUMMARY OF THE INVENTION

These and other objects are achieved by the present invention which isdirected to a vaccine for prevention and treatment of infection bygram-negative bacteria, and a method of immunizing poultry and otheranimals against such infections using the vaccine. The invention alsoprovides a method for isolating and purifying outer membrane siderophorereceptor proteins from gram-negative bacteria for producing the vaccine.The invention further provides an in vitro method of diagnosinginfections of gram-negative bacteria in an animal using antibodiesraised to the isolated receptor proteins.

The vaccine is useful for immunizing an avian or other animal againstinfection by gram-negative bacteria such as colibacillosis,salmonellosis and pasteurellosis. The vaccine is composed of asubstantially pure siderophore receptor protein derived from the outermembrane of a gram-negative bacteria, for example, Salmonella spp.,Escherichia spp. and Pasteurella spp. A siderophore receptor protein,useful according to the invention, is a protein or antigenic peptidesequence thereof derived from the outer membrane of a gram negativebacterium, which is capable of producing an antibody that will reactwith the siderophore receptor protein expressed by a gram-negativebacteria of the same or different strain, species or genus. Preferably,the siderophore receptor protein is derived from a bacterium belongingto the family Enterobacteriaceae and/or Pasteurellaceae.

The vaccine contains siderophore receptor proteins (SRPs) derived from agram-negative bacteria, capable of eliciting an immune response in ananimal with the production of anti-SRP antibodies. These antibodies willreact with siderophore receptor proteins of that bacteria, and may alsocross-react with siderophore receptor proteins of a different strain,species and/or genera of gram-negative bacteria to providecross-protection against infection from such other bacteria. Usefulsiderophore receptor proteins having a molecular weight of about 72-96kDa, as determined by SDS-PAGE, have been isolated from E. coli,Salmonella spp., Pasteurella spp., Pseudomonas spp., and Klebsiella spp.Preferably, the siderophore receptor proteins (SRPs) are derived fromEscherichia coli, Salmonella spp. and/or Pasteurella spp. The antibodiesproduced from those SRPs will react with SRPs of those bacteria andcross-react with SRPs of a different strain, species and/or genera ofbacteria within the family Enterobacteriaceae and/or Pasteurellaceae.

The vaccine contains one or more siderophore receptor proteins extractedfrom the outer membrane of a single strain or species, or two or moredifferent strains or species of gram-negative bacteria. The amount andtype of siderophore receptor protein included in the vaccine iseffective to stimulate production of antibodies reactive with asiderophore receptor protein of one, preferably two or more strains,species or genera of gram-negative bacteria. A preferred vaccine iscomposed of an amount and profile of siderophore receptor-proteins toeffectively induce antibodies reactive with a majority, preferably all,of the siderophore receptor proteins of a bacterial population toeffectively enhance opsonization and complement-mediated bacteriallysis, and/or block the iron binding capacity of the bacteria. Thesiderophore receptor protein is combined with aphysiologically-acceptable carrier, preferably a liquid. The vaccine mayfurther include an adjuvant to enhance the immune response, and otheradditives as desired, such as preservatives, flavoring agents, bufferingagents, and the like.

The present invention also provides a method for isolating highquantities of immunogenically effective siderophore receptor proteinsfrom outer membranes of a single strain or species of gram-negativebacteria such as E. coli, Salmonella and/or Pasteurella. The methodincludes culturing the organism under conditions of low ironavailability, that is, in a culture medium that lacks iron or includesan iron chelating agent. The siderophore receptor proteins are thenseparated from the bacterial outer membrane and purified by use of theanionic detergent, sodium dodecyl sulfate, preferably under non-reducingconditions.

The siderophore receptor proteins may be utilized to raise polyclonalantibody sera and monoclonal antibodies for use in passive immunizationtherapies. Such antibodies may also be used in an in vitro method ofdiagnosing a gram-negative bacterial infection in an animal. Thediagnostic method includes contacting a body material potentiallyinfected with a gram-negative bacteria, such as a tissue sample or bodyfluid, with a labelled antibody raised to a siderophore receptorprotein, and detecting the label in the complex formed between thesiderophore receptor protein in the body material and the labelledantibody. The method may also be performed by combining the body samplewith the antibody to the siderophore receptor protein, and thencontacting the sample with a labelled anti-species antibody reactivewith the protein-specific antibody, and then detecting the label.

The siderophore receptor proteins can also be used as capture antigensin a method of monitoring and profiling gram negative sepsis. Forexample, the protein may be used in an ELISA technique In which theprotein is bound to a solid support and contacted with a sample materialto react with and detect antibodies present in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic depiction of the elution profile of concentrated,solubilized siderophore receptor proteins isolated from Escherichia coliserotype 078 (ATCC 55652).

FIG. 2 is a graphic depiction of the quantitative clearance ofSalmonella agona in spleens of turkeys vaccinated with IROMPs isolatedfrom E. coli and non-vaccinated controls.

FIG. 3 is a graphic depiction of the serological response to E. colisiderophore receptor proteins (SRPs) between vaccinated andnon-vaccinated flocks.

FIG. 4 is a depiction of the total % mortality and culls in control andE. coli SRP-vaccinated flocks (3-13 weeks of age).

FIG. 5 is a depiction of the total % mortality and culls in control andE. coli SRP-vaccinated flocks (3-13 weeks of age).

FIG. 6 is a graphic depiction of the total mortality in SRP-vaccinatedand non-vaccinated turkeys following challenge with Pasteurellamultocida P-1059.

FIG. 7 is a graphic depiction of the serological response in birdsvaccinated with purified siderophore receptor proteins from Salmonellasenftenberg, showing cross-reactivity with the SRP of E. coli.

FIG. 8 is a graphical depiction of the serological response in birdsvaccinated with purified siderophore receptor proteins from P.multocida, showing cross-reactivity with the SRP of E. coli.

FIG. 9 is a graphic depiction of the total % mortality in consecutiveflocks before and after vaccinating with siderophore receptor proteinsderived from E. coli 078.

FIG. 10 is a graphic depiction of the serological response to SRPs fromE. coli between SRP-vaccinated and non-SRP-vaccinated commercial turkeyflocks.

FIG. 11 is a graphical depiction of the serological response of purifiedSRP and whole cell of Salmonella heidelberg.

FIG. 12 is a graphic depiction of the total mortality between progeny ofSRP-vaccinated and non-vaccinated (control) breeder hens.

FIG. 13 is a graphical depiction of the serological response in birdsvaccinated with purified siderophore receptor proteins from Salmonellatyphimurium, showing cross-reactivity with the SRP of E. coli.

FIG. 14 is a graphical depiction of the serological response in birdsvaccinated with purified siderophore receptor proteins from Salmonellaenteritidis, showing cross-reactivity with the SRP of E. coli.

FIG. 15 is a graphical depiction of SRPs of Salmonella typhimurium as aprotective immunogen against a homologous and heterologous challenge inturkeys.

FIG. 16 is a graphical depiction of SRPs of Salmonella enteritidis asprotective immunogens against a homologous and heterologous challenge inturkeys.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “substantially pure” means that the siderophorereceptor protein has been extracted and isolated from its naturalassociation with other proteins, lipids, and other like substances andelements of a bacterial cell or other organism.

Gram-negative bacteria are frequent pathogens of poultry and otheranimals, such as domestic foul, livestock, horses, companion animals,and humans. In an iron-restricted environment, bacteria such asEscherichia coli, Salmonella spp. and Pasteurella spp. producesiderophores that chelate ferric iron and bind to outer membraneproteins that function as siderophore receptors on the bacterialmembrane.

The invention provides an improved process for isolating and separatingsiderophore receptor proteins from the outer membrane of gram-negativebacteria. Isolation and purification of immunogenically intactsiderophore receptor proteins from bacterial membranes in a sufficientquantity and immunogenic quality for formulating a vaccine againstinfection by gram-negative bacteria has been difficult. The structuralorientation, or conformation, of the outer membrane protein necessary toprovide antigenicity may be typically lost when the protein is separatedand purified from the lipopolysaccharide complex. Another problem isthat the protein becomes denatured by the separation process wherein itsimmunogenicity is lost. According to the present invention, however, theisolation and separation of immunogenic quantities of antigenicallyeffective siderophore receptor proteins from the outer membrane ofgram-negative bacteria has been achieved. This enables the production ofvaccines and hyperimmunized sera for the treatment of animals infectedor susceptible to infection by gram-negative bacteria, and in vitrodiagnostic methods for detecting such an infection in an animal.

As a group, gram-negative bacteria possess a common cell wall structure.Components of the cell wall structure may be used as immunogens.However, these immunogens may provide only homologous immune protection.The present vaccine utilizes a combination of outer membrane siderophorereceptor proteins common to two or more gram-negative bacteria that arecapable of proliferating in the blood or host tissues and causinginfection in an animal. The vaccine may contain two or more siderophorereceptor proteins (SRPs), preferably four or more SRPs derived from theouter membrane of one or more strains or species of gram-negativebacteria and/or other organism. Preferably, the SRPs are derived from asingle strain or species of gram-negative bacteria. A preferredsiderophore receptor protein for use in the vaccine has a commonreceptor reactive with siderophores produced by two or more strains,species and/or genera of gram-negative bacteria.

An example of a useful siderophore receptor protein is the receptorprotein for aerobactin (MW about 72-74 kDa) produced by members of thefamily Enterobacteriaceae, for example, Escherichia coli, Salmonella andKlebsiella. Antibodies produced against an aerobactin receptor proteinof one species, strain or genus of that family have been found tocross-react with other bacteria within the family. Species ofPseudomonas of the family Pseudomonadaceae also express aerobactinsiderophore receptor proteins that can be isolated according to theinvention and used in a vaccine to produce antibodies that cross-reactwith the aerobactin receptor proteins of E. coli, Salmonella andKlebsiella, among other members of the family Enterobacteriaceae.

Another example of a suitable siderophore receptor protein for use inthe present vaccines is that produced by Pasteurella multocida for thesiderophore multocidin (MW about 500-1000 kDa). Antibodies to themultocidin receptor protein will react with all three of the SRPs inPasteurella multocida. In Western blots, two of the larger siderophoreproteins (96 kDa, 84 kDa) of P. multocida showed reactivity withhyperimmune E. coli protein antisera. Antibodies produced to multocidinreceptor proteins will cross-react with the siderophore receptorproteins of Salmonella spp. and E. coli, as demonstrated by ELISA andWestern blot analysis.

Other siderophore receptor proteins include those reactive with thesiderophore enterochelin (MW about 81-84 kDa) produced by E. coli,Salmonella, Pseudomonas and Klebsiella; and the siderophore citrate (MWabout 74-78 kDa) produced by E. coli, among others. A vaccine containingthe enterochelin and/or citrate receptor proteins will produceantibodies reactive with E. coli, Salmonella and other bacteria of thefamily Enterobacteriaceae, and with Pseudomonas of the familyPseudomonadaceae.

Another useful SRP is the siderophore receptor protein for ferrichrome(MW about 78 kDa) produced by E. coli, and Salmonella spp. In commercialpoultry raising facilities, infection by Aspergillus causes seriousrespiratory problems in the birds. In the lungs, Aspergillus willexcrete ferrichrome to acquire iron as a growth nutrient. Under ironrestriction or systemic conditions, E. coli and Salmonella will expressferrichrome receptor protein. They are also opportunistic bacteria thatcan scavenge and utilize ferrichrome produced by Aspergillus as a growthnutrient. Therefore, it is preferred that the vaccine preparationinclude a ferrichrome receptor protein to induce antibodies that willbind and cross-react with the ferrichrome receptor proteins ofgram-negative bacteria including E. coli and Salmonella, and fungi/mold.A vaccine containing this SRP will elicit an immune response to theprotein to enhance the bactericidal activity of the antibody. Also, oncethe avian or other animal is vaccinated with a ferrichrome receptorprotein, Aspergillus expressing this protein in vivo in the animal willenhance the antibody response to the ferrichrome receptor protein whichin turn will cross-react with Salmonella and E. coli and other bacteriathat express the ferrichrome receptor protein.

Antibody elicited from a ferrichrome receptor protein (MW about 78 kDa)derived from E. coli can cross-react with the receptor proteins offungi, such as Aspergillus flavus, Aspergillus fumigatus, Penicilliumand Fusarium. Western blot analysis against the outer membrane proteins(OMPs) of A. fumigatus using anti-SRP antibody revealed threecross-reactive proteins (MW about 45-90 kDa). The inclusion of aferrichrome receptor protein into a vaccine preparation will provideinducement of antibodies that will react with the fungi and/or bacteriato prevent binding and excretion of the ferrichrome siderophore. Animalssuch as birds that are vaccinated with a vaccine preparation containinga ferrichrome receptor protein will get an elevated antibody titer bybacteria and/or fungi that challenge the animal and produce aferrichrome receptor protein. Also, antibody to the ferrichrome receptorcan be elevated by natural field challenge by bacteria or fungi whichcan induce a bactericidal effect that could lessen system challenge anddisease potential.

Yet another useful SRP is a coprogen receptor protein (MW about 74-76kDa) produced by E. coli. Antibodies produced against coprogen receptorprotein will cross-react with the SRPs of other E. coli expressing thisprotein under systemic conditions.

In one embodiment, the vaccine is formulated with siderophore receptorproteins (SRPs) of different types and/or molecular weights, derivedfrom a first gram-negative bacteria, the SRPs being capable ofstimulating production OL antibodies that react with the firstgram-negative bacteria as well as a second gram-negative bacteria of adifferent strain or species than the first gram-negative bacteria. Thevaccine preferably contains all SRPs derived from the gram-negativebacteria infectious agent. For example, P. multocida and Salmonella spp.have been identified as producing 3 SRPs each, and E. coli produced 2,3, 4, and 6 SRPs varying between serotypes. Accordingly, the vaccine isformulated to contain the SRPs derived from the bacterial causativeagent, i.e., 2-6 or more SRPs. It is preferred that the vaccine alsoinclude siderophore receptor proteins of different types and/ormolecular weights derived from a gram-negative bacteria of a strain orspecies different than the first gram-negative bacteria, preferably 1-15SRPs, preferably 5-10 SRPs.

For example, the vaccine may contain a siderophore receptor proteinderived from E. coli, preferably E. coli serotype 01a, 02a and/or 078,that is capable of stimulating production of an antibody immunoreactivewith that E. coli and a second gram-negative bacteria such as Salmonellaspp., Pseudomonas aeruginosa, Klebsiella pneumoniae and/or Pasteurellamultocida. In another example, the vaccine may contain a siderophorereceptor protein derived from a species of Pasteurella, such as P.multocida, that is capable of stimulating production of an antibodyimmunoreactive with that species of Pasteurella and a secondgram-negative bacteria such as Salmonella spp. and/or E. coli. In yetanother example, the vaccine may contain a siderophore receptor proteinderived from a species of Salmonella that is capable of stimulatingproduction of an antibody immunoreactive with that species of thatspecies of Salmonella, and a second gram-negative bacteria such as E.coli, Pseudomonas, Klebsiella, and/or Pasteurella multocida.

A vaccine formulated with siderophore receptor proteins derived from E.coli is preferably composed of an aerobactin, ferrichrome, coprogen,enterochelin and/or citrate SRP, having molecular weights of about 89kDa to about 72 kDa, as determined by SDS-PAGE. The vaccine preferablyincludes 2-5 receptor proteins, preferably 3-5 proteins, preferably allfive E. coli SRPs. A preferred vaccine against E. coli infection isprepared with the SRPs from E. coli 078 (ATCC #55652). E. coli 078 hasbeen identified as producing up to 6 SRPs ranging in molecular weightfrom about 72 to 90 to 92 kDa, as determined by SDS-PAGE. The SRPsderived from E. coli 078 include aerobactin, ferrichrome, coprogen,enterochelin and citrate SRPs, having molecular weights of about 91-92kDa, 89 kDa, 84 kDa, 78 kDa, 74 kDa and 72 kDa, as determined bySDS-PAGE, 12.5% acrylamide reducing gel. Although the 91-92 kDa proteinsof E. coli 078 are expressed in culture media made with and withoutiron, the expression of those proteins is enhanced in an iron-restrictedmedium, and as used herein, the 91-92 kDa proteins are considered to beiron-regulated SRPs. A preferred vaccine for immunizing an animalagainst E. coli is formulated with an aerobactin, ferrichrome, coprogen,enterochelin and citrate SRP derived from E. coli, preferably E. coli078, made of at least 5 siderophore receptor proteins, preferably atleast 6 receptor proteins, or more, to induce anti-SRP antibodies toeffectively block a majority, preferably all, iron binding sites of E.coli serotypes present in an infection, and to induce high antibodylevels to promote bactericidal activity.

It is further preferred that the vaccine includes one or more SRPs,preferably about 1-15 SRPs, derived from one or more additionalbacteria, different from the first gram-negative bacteria. For example,in a vaccine composed of SRPs from E. coli, it is desirable to includeone or more of the SRPs derived from Salmonella, Pasteurella multocida,Klebsiella and/or Pseudomonas.

A preferred vaccine contains each of the SRPs of different types and/ormolecular weights, of a population of gram-negative bacteria to induceproduction of antibodies that will effectively block the iron-bindingsites of all of the various SRPs of the bacterial population so that thebacteria cannot effectively bind iron as a nutrient for growth. It isalso preferred that the vaccine will induce high SRP antibody levelsthat will enhance opsonization and/or complement-mediated bacteriallysis. Due to the variation in iron-regulated outer membrane proteins(IROMPs) produced between and within bacterial serotypes, formulating avaccine with SRPs isolated and purified from a single isolate source mayprovide only a partial profile of the SRPs present in a bacterialpopulation. Consequently, the effectiveness of the vaccine to induceanti-SRP antibodies to block bacterial iron-binding sites and inhibitbacterial infection may be limited to those serotypes that produce allor less than all of the SRPs included in the vaccine, while thosebacterial serotypes producing other SRPs may retain an iron-bindingcapacity. Thus, it is preferred that a profile, or banding pattern(i.e., SDS-PAGE protein separations), of a bacterial population isconducted by examining different field isolates, preferably about 25-100isolates, to determine the SRPs that are present, and all of the variousSRPs are included in the vaccine.

Non-iron regulated proteins and polypeptides may also be included in thevaccine as adjuvants to enhance the effectiveness of the vaccine andincrease opsonization, that is, increase macrophage activity resultingin increased phagocytosis of antibody-bound cells, and inducecomplement-mediated bacterial lysis. A useful adjuvant protein is a34-38 kDa group of outer membrane proteins (porins, i.e., pore-formingproteins) derived from gram-negative bacteria of the familyEnterobacteriaceae and Pasteurellaceae including E. coli 078, and othergram-negative bacteria. The transmembrane and porin proteins (MW 34-38kDa) identified as OmpA, OmoC, OmpD and OmpF are expressed with andwithout iron, are relatively conserved between gram-negative bacteria,and play a role in iron binding. For example, OmpF and OmoC will bindlactoferrin (Erdei et al., Infection and Immunity 62:1236-1240 (April1994)), while OmpA will bind ferrichrome (Coulton et al., J. Gen.Microbiol. 110:211-220 (1979)). Antibodies early in infectionparticularly of the IgM class will cross-react with outer membraneproteins of E. coli, Salmonella, Pasteurella, Pseudomonas andKlebsiella, and will bind lactoferrin and/or ferrichrome, precluding theavailability of an iron source for bacterial growth. Antibodies to theseproteins will also bind to the porin Omp on the surface to enhanceopsonization and/or complement-mediated bacterial lysis. Immunogenicallyintact 34-38 kDa porin outer membrane proteins can be isolated andpurified according to the process of the invention.

The vaccine may be used to immunize poultry and other animals such asdomestic fowl, livestock, horses, companion animals, and humans, againstinfection caused by one or more gram-negative bacteria. The vaccine iseffective for eliciting antibodies that are immunoreactive with agram-negative bacteria that expresses one or more siderophore receptorprotein(s).

Preferably, the vaccine is capable of achieving clinical efficacy ofcross-reactive and cross-protective immunization against two or moredifferent strains, species and/or genera of gram-negative bacteria orother organisms capable of expressing siderophore receptor proteins. Forexample, a vaccine containing siderophore receptor proteins foraerobactin, enterochelin, ferrichrome, coprogen and/or citrate, may beused to stimulate production of antibodies that cross-react with anumber of different bacteria that express one or more of these receptorproteins. The effectiveness of the present vaccine is due, at least inpart, to the conservative nature of the outer membrane siderophorereceptor proteins which are cross-reactive with siderophore receptorproteins produced by two or more different species, strains and/orgenera of Enterobacteriaceae such as E. coli, Salmonella, and othergram-negative bacteria within other families such as Pasteurella and/orPseudomonas.

Because of the cross-reactivity of the SRPs, the vaccine is effective instimulating production of a antibodies that react with the firstgram-negative bacteria (from which the SRPs were derived), as well as asecond gram-negative bacteria of a different strain or species than thefirst gram-negative bacteria. For example, a vaccine can be formulatedto contain a siderophore receptor protein derived from E. coli,preferably E. coli serotype 01a, 02a and/or 078, more preferably E. coli078, that is effective in stimulating production in vivo of an antibodyimmunoreactive with that E. coli serotype (from which the SRP(s) werederived), and a second gram-negative bacteria such as Salmonella spp.,Pseudomonas aeruginosa, Klebsiella pneumoniae and/or Pasteurellamultocida. In another example, the vaccine can contain a siderophorereceptor protein derived from a species of Pasteurella, such as P.multocida, that is effective in stimulating production of an antibodyimmunoreactive with that species of Pasteurella and a secondgram-negative bacteria such as Salmonella spp. and/or E. coli. In yetanother example, the vaccine can contain a siderophore receptor proteinderived from a species of Salmonella that is effective in stimulatingproduction of an antibody immunoreactive with that species ofSalmonella, and a second gram-negative bacteria such as E. coli,Pseudomonas, Klebsiella, and/or Pasteurella multocida.

Advantageously, immunization using the present vaccine containing animmunogen cross-reactive with multiple species, strains and genera ofgram-negative bacteria, not only minimizes immunization costs sinceseparate inoculations with a different immunogen for each type ofgram-negative bacteria is not required. In addition, the present vaccineprovides protection against new strains or unanticipated pathogens ofgram-negative bacteria which produce siderophore receptor proteins thatwill cross react with antibodies induced by the siderophore receptorproteins contained in the vaccine. The vaccine given to an adult animalis highly efficacious in treating and preventing gram-negative sepsisnot only in the adult animal but also their progeny by the directtransfer of anti-SRP antibodies.

Commercial bacterial whole cell vaccines are useful for treating aparticular disease and/or infection but do not provide effectivecross-protection against other infection. For example, avianpasteurellosis in turkeys caused by Pasteurella multocida is clinicallydiagnosed by particular lesions induced by the bacterial infection.Treating the disease with a commercial whole cell vaccine stimulatesantibodies that are homologous but not heterologous in their action, andwill not cross-protect against infection by other bacteria.

Advantageously, the present vaccines provide cross-protection against anumber of infections caused by gram-negative bacteria. According to theinvention, an animal species suffering from gram-negative bacterialsepsis can be administered the vaccine containing SRPs derived from the(causative agent) gram-negative bacteria to induce antibodiesimmunoreactive with those SRP(s) to inhibit the disease state. Theantibodies will also cross-react with SRP(s) produced by anothergram-negative bacteria to inhibit a disease state caused by that otherbacteria. Thus, a vaccine containing SRPs of a first gram negativebacteria will provide protection against an infection caused by thatbacteria and provide cross-protection against infection caused by adifferent gram-negative bacteria.

Gram-negative bacteria suitable for use in obtaining siderophorereceptor proteins according to the invention, are those capable ofproducing siderophore receptor proteins when raised under growthconditions of low iron availability. Examples of useful gram-negativebacteria include Escherichia coli (serotypes 01a, 02a, and 078),Salmonella agona, Salmonella blockley, Salmonella enteriditis,Salmonella hadar, Salmonella Heidelberg, Salmonella montevideo,Salmonella senftenberg, Salmonella cholerasuis, Salmonella typhimurium,Pasteurella multocida (serotype A:3,4), Klebsiella pneumoniae,Pseudomonas aeruginosa, and the like These organisms are commerciallyavailable from a depository such as American Type Culture Collection(ATCC), Rockville, Md. In addition, such organisms are readilyobtainable by isolation techniques known and used in the art. Thegram-negative bacteria may be derived from an infected animal as a fieldisolate, and screened for production of SRPs, and introduced directlyinto the preferred iron-depleted media for that bacteria, or stored forfuture use, for example, in a frozen repository at about −20° C. toabout −95° C., preferably about −40° C. to about −50° C., in BHIcontaining 20% glycerol, and other like media.

For producing the siderophore receptor proteins, conditions of low ironavailability are created using culture media that lack iron or have beensupplemented with an iron chelating agent to decrease iron availability.Suitable culture media for providing low iron availability and promotingproduction of the siderophore receptor proteins in gram-negativebacteria, include media such as tryptic soy broth (Difco Laboratories,Detroit, Mich.) and/or brain-hear infusion (BHI) broth which has beencombined with an ion-chelating agent, for example, α,α′-dipyridyl,deferoxamine, and other like agents. In a preferred embodiment,α,α-dipyridyl is added to a BHI culture media in a concentration ofabout 1-500 μg/ml, preferably about 50-250 μg/ml, more preferably about75-150 μg/ml.

The gram-negative bacteria employed to produce a siderophore receptorprotein are cultured in the preferred media for that organism usingmethodologies and apparati known and used in the art, such as afermenter, gyrator shaker, or other like apparatus. For example, aculture may be grown in a gyrator shaker in which the media is stirredcontinuously with aeration at about 300-600 rev/minute, for about 15-20hours, at a temperature and pH appropriate for growth for that organism,i.e., about 35-45° C. and about pH 7-7.6, preferably pH 6.5-7.5. Thebacterial culture is then processed to separate and purify thesiderophore receptor proteins from the outer membrane of the bacteria.

The bacterial culture is concentrated, for example, by centrifugation,membrane concentration, and the like. For example, the cell culture maybe centrifuged at about 2,450-20,000×g, preferably at about5,000-16,000×g, for about 5-15 minutes at about 3-6° C. The supernatantis removed by decanting, suctioning, pipetting and the like, and theconcentrated cell pellet is collected and washed in a compatible buffersolution maintained at about pH 7-7.6, such as tris-buffered saline(TBS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),3-N(N-morpholino)propanesulfonic acid (MOPS), and the like. The washedpellet is resuspended and washed in a compatible buffer solution, i.e.,TBS, HEPES, MOPS and the like. The cell material is then treated tosolubilize the components of the outer membrane by resuspending thepellet in buffer containing about 0.5-10% sodium N-lauroyl sarcosinate,preferably about 1-3%, at about 4-10° C. for about 15 minutes to about 3hours, preferably about 30 minutes to about 2 hours, preferably withcontinuous stirring.

The bacterial cells are then disrupted by sonication, French pressure,grinding with abrasives, glass bead vortexing, and other like methodsknown and used in the art, preferably at a temperature of about 3-6° C.The cell homogenate is then centrifuged at about 10,000-20,000×g forabout 10-45 minutes, to separate cell debris from the supernatantfraction containing the outer membrane proteins. The supernatant iscollected by decanting, suctioning, pipetting, or other like method, andthen concentrated, for example, by ethanol precipitation, membraneconcentration, propylene glycol precipitation, and other methods knownand used in the art. In a preferred method, the supernatant is treatedby passing it through a membrane having a molecular weight cut-off ofabout 1,000-50,000 MW, preferably about 10,000-25,000 MW, to concentratethe protein and allow contaminating proteins smaller than the molecularweight cut-off to pass through the membrane, and to decrease the amountof detergent. Such membranes are commercially available, for example,from Amicon, Danvers, Mass.

The concentrated supernatant is then reconstituted in a compatiblebuffer, i.e., TBS, HEPES, MOPS, and the like, about pH 7-7.6, whichcontains a detergent for solubilizing the outer membrane and extractingthe siderophore receptor proteins. It was found that the anionicdetergent sodium dodecyl sulfate (SDS), when used as a solubilizingdetergent alone without a reducing agent such as 2-mercaptoethanol, isparticularly effective for extracting a high quantity of the siderophorereceptor proteins without denaturing or altering their immunogenicitysuch that the proteins will function in vivo as effective immunogens toelicit an antibody response against gram-negative bacteria. The buffersolution contains about 0.1-4% SDS (0.2%), preferably about 0.1-2% SDS,preferably about 0.1-2% SDS.

After about 1-10 minutes, the siderophore receptor proteins areseparated from the buffer solution by affinity, ion exchange, sizeexclusion and other like chromatographic methods known and used in theart. Preferably, the SRP preparation is separated with a 4% stacking gelon a 12.5% acrylamide reducing gel. The fractions are then combined,concentrated, for example by centrifuging, and precipitated, for examplewith an alcohol (i.e., ethanol, methanol, acetone), to remove the SDS.The purified proteins may be used immediately to prepare a vaccine, ormay be stored for future use through lyophilization, cryopreservation,or other like technique known and used in the art.

The vaccine of the present invention may be used for preventing-andeliminating infections of gram-negative bacteria in poultry and otheranimals, including humans. The vaccine may be delivered to the animal,for example, by parenteral delivery, injection (subcutaneous orintramuscular), sustained-released repository, aerosolization, egginoculation (i.e., poultry), and the like, by known techniques in theart. For prophylactic and anti-infectious therapeutic use in vivo, thevaccine contains an amount of a siderophore receptor protein tostimulate a level of active immunity in the animal to inhibit and/oreliminate gram-negative bacterial pathogenesis and/or sepsis.

The siderophore receptor proteins are administered in combination with apharmaceutical carrier compatible with the protein and the animal.Suitable pharmacological carriers include, for example, physiologicalsaline (0.85%), phosphate-buffered saline (PBS), Tris(hydroxymethylaminomethane (TRIS), Tris-buffered saline, and the like. The protein mayalso be incorporated into a carrier which is a biocompatible and canincorporate the protein and provide for its controlled release ordelivery, for example, a sustained release polymer such as a hydrogel,acrylate, polylactide, polycaprolactone, polyglycolide, or copolymerthereof. An example of a solid matrix for implantation into the animaland sustained release of the protein antigen into the body is ametabolizable matrix, as described, for example, in U.S. Pat. No.4,452,775 (Kent), the disclosure of which is incorporated by referenceherein.

Adjuvants may be included in the vaccine to enhance the immune responsein the animal. Such adjuvants include, for example, aluminum hydroxide,aluminum phosphate, Freund's Incomplete Adjuvant (FCA), liposomes,ISCOM, and the like. The vaccine may also include additives such asbuffers and preservatives to maintain isotonicity, physiological pH andstability. Parenteral and intravenous formulations of the vaccine mayinclude an emulsifying and/or suspending agent, together withpharmaceutically-acceptable diluents to control the delivery and thedose amount of the vaccine.

Factors bearing on the vaccine dosage include, for example, the age andweight of the animal. The range of a given dose is about 25-5000 μg ofthe purified siderophore receptor protein per ml, preferably about100-1000 μg/ml preferably given in about 0.1-5 ml doses. The vaccineshould be administered to the animal in an amount effective to ensurethat the animal will develop an immunity to protect against agram-negative bacterial infection. For example, for poultry, a singledose of a vaccine made with Freund's Incomplete Adjuvant would containabout 150-300 μg of the purified siderophore receptor protein per ml.For immunizing a one-day of age bird of about 60 grams weight, the birdmay be subcutaneously or intramuscularly injected with an about 0.25-0.5ml dose. For an about 3-week old bird of about 1.5 lbs, the bird may beinjected with about 0.25-1 ml dose. A vaccine for immunizing an about5-lb piglet against Salmonella cholerasuis would contain about 100-5000μg protein per ml, preferably given in 1-5 ml doses. In each case, theimmunizing dose would then be followed by a booster given at about 21-28days after the first injection. Preferably, the vaccine is formulatedwith an amount of the siderophore receptor protein effective forimmunizing a susceptible animal against an infection by two or morestrains or species of gram-negative bacteria that express a siderophorereceptor protein.

For boosting the immunizing dose, the booster may be a preparation ofwhole cells as conventionally used, or a chemically modified cellpreparation, among others. For example, a useful booster is apreparation of a modified E. coli such as avirulent R-mutants, as forexample, E. coli J5 (commercially available from ATCC as ATCC #43754;described by Overbeck et al., J. Clin. Microbiol. 25:1009-1013 (1987)),or Salmonella minnesota (commercially available from ATCC as ATCC#49284; as described by Sanderson et al., J. Bacteriol. 119:753-759,760-764 (1974)) that lack outer oligosaccharide side chains of thelipopolysaccharide (LPS) layer of the outer membrane. Outeroligosaccharide side chains tend to mask SRPs on the cell membrane insuch a way that the immune system does not recognize the SRPs andanti-SRP antibody titers are depressed. To enhance the ability of abooster made with intact bacterial cells to elicit an anti-SRP immuneresponse, the cell membrane of the bacteria can be chemically altered toeliminate the interfering oligosaccharide side chains. Boosting withchemically-modified bacteria such as an R-mutant, advantageouslyprovides an anti-SRP antibody titer that is 5-20 times higher thanbooster made of a non-modified whole cell bacterial preparation, or anatural field challenge.

Although not intended as a limitation of the invention, the mechanism bywhich immunization with the present vaccine provides protection againstgram-negative bacterial infection is believed to be as follows. After ananimal has been immunized with the vaccine, upon being challenged with apathogenic strain of gram-negative bacteria, the body responds byproducing humoral antibodies that block the siderophore receptorproteins on the outer membrane of the bacteria. This prevents ironuptake by the cell, which, in turn, eventually starves the bacteria ofrequired iron nutrients. Another mechanism is that humoral antibodiesproduced in response to the siderophore receptor proteins in thevaccine, bind to the siderophore receptor protein on the bacterialmembrane to cause activation of compliment (C′). This results incomplement-mediated bacteriolysis, or increased opsonization which leadsto increased phagocytosis by the mononuclear phagocytic system.

In addition, the efficacy of this vaccine is based on the use ofpurified siderophore receptor proteins rather than using whole cells.The immune response in animals vaccinated with a purified SRPpreparation is about 20 times greater than the immune response to apreparation of whole cell grown under iron-restricted conditions. Duringgram-negative sepsis, an animal host mounts an immune response to aninvading bacteria. Since the major constituent of the cell wall ofgram-negative bacteria is made of lipopolysaccharide (LPS), the immuneresponse of an animal is directed to this structure inducing animmunodominant role for the LPS cell wall. Outer membrane proteins suchas IROMPs or SRPs that are not dominant proteins on the surface of thebacterial cell wall induce limited immune response resulting in lowantibody titers. Thus, the use of a bacterin made of whole bacterialcells grown under iron restriction to express siderophore receptorproteins provides a limited immune response to the siderophore receptorproteins due to competing antigens on the cell surface. By comparison,immunizing an animal with a vaccine made of purified SRPs, there is lessantigenic competition and the animal's immune system focuses itsresponse on the receptor proteins Serological profiles show asignificant increase in antibody titer in the SRP-vaccinated groupcompared to the whole cell-vaccinated group when boosted with whole cellexpressing SRP.

Polyclonal antibodies may be raised to the siderophore receptor proteinby hyperimmunizing an animal with an inoculum containing the isolatedsiderophore receptor protein. The blood serum may be removed andcontacted with immobilized siderophore receptor proteins reactive withthe protein-specific antibodies. The semi-purified serum may be furthertreated by chromatographic methods to purify IgG and IgM immunoglobulinsto provide a purified polyclonal antibody sera for commercial use.

Monoclonal antibodies reactive with the siderophore receptor protein maybe raised by hybridoma techniques known and used in the art. In brief, amouse, rat, rabbit or other appropriate species may be immunized with asiderophore receptor protein. The spleen of the animal is then removedand processed as a whole cell preparation. Following the method ofKohler and Milstein (Nature 256:496-97 (1975)), the immune cells fromthe spleen cell preparation can be fused with myeloma cells to producehybridomas. The hybridomas may then be cultured and the culture fluidtested for antibodies specific for siderophore receptor proteins using,for example, an ELISA in which specific siderophore receptor proteinsare attached to a solid surface and act as capture antigens. Thehybridoma may then be introduced into the peritoneum of the host speciesto produce a peritoneal growth of the hybridoma, and ascites fluidscontaining the monoclonal antibody to the bacteria may be collected.

The monoclonal antibodies may be used in diagnostic and therapeuticcompositions and methods, including passive immunization. For example,immunoglobulins specific towards a siderophore receptor protein may beused to provide passive immunity against gram negative sepsis. Animalsmay be treated by administering immunoglobulins intramuscularly at about100/mg/kg body weight, about every 3-7 days.

A method for diagnosing an infection by gram-negative bacteria in a bodysample may be carried out with the polyclonal antibody sera ormonoclonal antibodies described hereinabove, in an enzyme-linkedimmunosorbant assay (ELISA), radioimmunoassay (RIA), immunofluorescentassay (IFA), a Northern, Western or Southern blot assay, and the like.In brief, the antibody or body sample (i.e., tissue sample, body fluid)may be immobilized, for example, by contact with a polymeric materialsuch as polystyrene, a nitrocellulose paper, or other like means forimmobilizing the antibody or sample. The other antibody or body sampleis then added, incubated, and the non-immobilized material is removed bywashing or other means. A labeled species-specific antibody reactivewith the later is added. The serum antibody or gram-negative bacteria inthe body sample, is then added and the presence and quantity of label isdetermined to indicate the presence and amount of gram-negative bacteriain the body sample.

The invention will be further described by reference to the followingdetailed examples, wherein the methodologies are as described below.These examples are not mean to limit the scope of the invention that hasbeen set forth in the foregoing description. Variation within theconcepts of the invention are apparent to those skilled in the art. Thedisclosures of the cited references throughout the application areincorporated by reference herein.

EXAMPLE 1 Production and Purification of Siderophore Receptor Proteins

Escherichia coli serotype 078 (turkey isolate; serotyped by PennsylvaniaState University, deposited with the American Type Culture Collection(ATCC), Bethesda, Md., U.S.A., as ATCC 455652, on Jan. 3, 1995) (700 mlat 10⁸ colonies/ml) was inoculated into a Virtis bench-top fermenter(Virtis, Inc., Gardiner, N.Y.), charged with 20-L of brain-heartinfusion (BHI, Difco Laboratories, Detroit, Mich.) containing 50μgrams/ml of dipyridyl (Sigma Chemical Co., St. Louis, Mo.) at 41° C.This isolate has been shown to produce four siderophore receptorproteins for (MW 89 kDa, 84 kDa, 78 kDa, 72 kDa) under iron-restrictiveconditions. The pH was held constant at 7.4 by automatic titration with5N NaOH. The fermenter was stirred at 400 rpm. The culture was growncontinuously for 18 hours after which the bacteria were removed bycontinuous-flow centrifugation at 20,000×g at 4° C. using a Beckman(Model J2-21M) centrifuge (Beckman Instruments, Eden Prairie, Minn.).The pelletized bacteria were washed two times with 1,000 mlphysiological saline (0.85%) to remove contaminating culture mediaproteins.

The bacteria were resuspended in tris-buffered saline (TBS) containing2.0% sodium N-lauroyl sarcosinate (SARKOSYL™, Sigma Chemical Co., St.Louis, Mo.), optical density 5%, 540 nm. The suspension was incubated at4° C. for 45 minutes with continuous stirring. The cells were thendisrupted using a continuous-flow cell sonicator (Banson 450, Danbury,Conn.) at 4° C., with a maximum flow rate of 5 gph. The disrupted cellsuspension was centrifuged at 16,000×g for 20 minutes.

The effluent from the continuous-flow cell sonicator containing theouter membrane proteins was collected and concentrated using ethanolprecipitation at −20° C. It is understood that the supernatant may alsobe concentrated by membrane concentration using a 50,000 MW cut offdiaflow membrane (Amicon, Danvers, Mass.). The concentrated material(10% T at 540 nm) was solubilized using 0.2 percent sodium dodecylsulfate (SDS) in TBS at pH 7.4.

The elution profile of the concentrated material treated with 0.2% SDSis shown in FIG. 1. The solubilized material was applied to a Vantagecolumn (Amicon, Danvers, Mass.) containing 3.2-L of cellufine fast flowGC-700 gel matrix (Amicon, Danvers, Mass.) equilibrated with TBScontaining 0.2% SDS at 25° C. Purification of the protein was monitoredby UV absorption at 280 nm. Flow rate through the column was 3,000 ml/hrand 15-ml fractions were collected using a UA-5 Detector and Retriever 5fraction collector (ISCO, Inc., Lincoln, Nebr.). Fractions from eachpeak were pooled and concentrated using a Diaflo ultrafiltrationapparatus with a 50,000 MWCO membrane. Concentrated material from eachpeak was examined by gel electrophoresis. As shown in FIG. 1, peak 1contained approximately 85% pure siderophore proteins. This solution wasethanol precipitated at −20° C. for 24 hours to remove the SDS, and thenresuspended in phosphate buffered saline (PBS). The amount of proteinwas determined using a Pierce BCA protein assay (Pierce, Rockford,Ill.).

EXAMPLE 2 Preparation of Vaccine with Siderophore Receptor Proteins

The precipitate from Example 1, hereinabove, containing siderophorereceptor proteins of E. coli serotype 078, were resuspended inphysiological saline (0.85%) containing 0.1% formalin as a preservative.The protein concentration was 300 μg/ml. The aqueous protein suspension(1,000 ml) was emulsified in a water-in-mineral oil adjuvant containing972 ml Drakeol 6 mineral oil and 28 ml of Anlacel A as an emulsifier.The mixture was emulsified using an Ultra-Turnax T50 emulsifier (KIKAWorks, Inc., Cincinnati, Ohio) at 4° C. The water-in-oil emulsion wasstored at 4° C.

Example 3 Vaccination of Poultry with Siderophore Receptor ProteinVaccine

Seventy-two turkey poults were raised in isolation from one day of age.At three weeks of age, the birds were divided into two equal groups.Group 1 was vaccinated subcutaneously with the vaccine from Example 2above, at a dosage level of 150 μg of siderophore receptor protein perbird. Group 2 remained as non-vaccinated controls. Group 1 was given abooster vaccination with the vaccine at a dosage level of 250 μgsiderophore receptor protein per bird at 18 days after the firstvaccination.

The vaccinated and non-vaccinated birds were equally divided among fourisolation rooms. Rooms A and B contained the vaccinated birds, and RoomsC and D contained the non-vaccinated controls. At seven weeks of age,birds in Groups A and C were challenged subcutaneously with Salmonellaagona at 1.0×10⁸ cfu/bird. At 24, 48, 72, 96 and 120 hourspost-challenge, two controls and two vaccinated birds were killed. Thespleens were aseptically removed from each bird and individuallyweighed, and adjusted to 4 crams/spleen, 10 grams/liver. Each sample wasthen homogenized in sterile physiological saline using a Stomacher LabBlender, Model 3500 (Seward Medical, London). Serial ten-fold dilutionsof each homogenate was plated in duplicate on brilliant sulfur greenplates (Difco Laboratories, Detroit, Mich.).

The results show the quantitative clearance of Salmonella agona inspleens of SRP-vaccinated and non-vaccinated turkeys (FIG. 2). Time 0represents the number of bacteria given to each bird. At 24-hourspost-challenge in the vaccinated birds, the level of bacteria werereduced to zero and remained at that level throughout the samplingperiod. In contrast, the non-vaccinated controls remained positive forthe duration of the experiment.

EXAMPLE 4 Cross-Reactivity of Siderophore IROMPS Produced by Escherichiacoli (Serotype 078)

Hyperimmunized serum produced against purified siderophore receptorproteins was examined for its cross-reactivity to bacteria fromdifferent genera and species. Siderophore receptor proteins wereproduced in the following bacteria: Escherichia coli (serotypes 01a, 02aand serotype 078 (ATCC #55652)), Salmonella agona, Salmonella blockley,Salmonella enteriditis, Salmonella hadar, Salmonella heidelberg,Salmonella montevideo, Salmonella senftenberg, Salmonella cholerasuis,and Pasteurella multocida (serotype A:3,4; deposited with ATCC as ATCC#______, on Feb. ______, 1995. These bacteria, except for S.cholerasuis, were field isolates obtained from clinically diagnosedbirds and serotyped by the State Poultry Testing Laboratory, Willmar,Minn. (Salmonella spp.) and Pennsylvania State University (E. coli).Salmonella cholerasuis was obtained from the University of MinnesotaDiagnostic Laboratory. The bacterial isolates were grown in 100 ml ofBHI broth with dipyridyl (175 mM), and without dipyridyl but containing200 μm ferric chloride.

The bacteria were collected from the cell cultures by centrifugation at16,000×g for 10 minutes at 4° C. The cell pellets were washed twice intris-buffered saline (TBS) at pH 7.4 and resuspended in 30 ml TBS. Thecells were ultrasonically disrupted for 2 minutes at 4° C. using aBranson Ultrasonic Sonicator (Danbury, Conn.). The disrupted cellsuspension was centrifuged at 160,000×g for 20 minutes at 4° C. Thesupernatant was collected centrifuged at 30,000×g for 2 hours at 4° C.The pellet was resuspended in 10 ml TBS containing 2% sodium n-lauroylsarcosine and placed on a gyratory shaker for 45 minutes at 4° C. Thedetergent insoluble outer membrane protein-enriched fraction wascollected by centrifugation at 30,000×g for 2 hours at 4° C. The pelletwas resuspended in 1 ml TBS and stored at −90° C. Proteins wereseparated by SDS-PAGE with a 4% stacking gel on a 12% resolving gel.Laemmli, U.K., Nature, 227:680-685 (1970).

The outer membrane proteins from the different E. coli, Salmonella andPasteurella isolates were transferred from the SDS-PAGE gels tonitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.). Themembranes were probed with negative (control) and positive antisera tothe siderophore receptor proteins.

The control antisera was collected from the birds in group 2, asdescribed in Example 3 hereinabove. The positive antisera was collectedfrom birds in group 1 from Example 3 hereinabove, at 5 days after thesecond vaccination. The sera, 50 ml each, were absorbed with killedwhole cell bacteria (E. coli 078, Salmonella heidelberg, Pasteurellamultocida) grown in iron-replete media (BHI containing 200 μm ferricchloride) for 1 hour at 4° C.

The SDS-PAGE patterns of the outer membrane protein extracts of thedifferent bacterial isolates, showed expression of siderophore receptorproteins when crown under conditions of iron restriction, in contrast tonon-iron restricted controls which did not express siderophore receptorproteins Pasteurella multocida produced three siderophore receptorproteins under conditions of iron restriction which had molecular massesof approximately 96 kDa, 84 kDa and 80 kDa The E. coli isolates producedslight variation in their IROMP profiles. Serotype 078 produced foursiderophore receptor proteins with approximate molecular mass of 89 kDa,84 kDa, 78 kDa and 72 kDa. Serotype 02a produced three bands withmolecular weights of 89 kDa, 78 kDa and 72 kDa. Serotype 01a producedtwo bands with molecular weights of 84 kDa and 78 kDa. All of theSalmonella isolates examined produced three siderophore receptorproteins with identical banding patterns with approximate molecularweights of 89 kDa, 81 kDa and 72 kDa.

Western blot analysis revealed that the positive antisera preparedagainst the purified siderophore receptor proteins of E. coli 078reacted intensely with the siderophore receptor proteins of E. coliserotypes 01a, 02a and the receptor proteins of Salmonella. The 96 kDaand 84 kDa receptor protein of Pasteurella reacted with the positive E.coli protein antisera. These results show that the siderophore receptorproteins of E. coli have complete antigenic homology to Salmonella andpartial homology to Pasteurella multocida. The control sera did notreact with any siderophore receptor proteins of those species.

EXAMPLE 5 Cross-Reactivity of Siderophore Receptor Proteins ofEscherichia Coli (Serotype 078)

Escherichia coli isolates (150 isolates) originating from colisepticemicbirds were screened for reactivity with the positive antisera of Example4, hereinabove. The isolates were examined by direct agglutination usingthe siderophore receptor antisera and negative reference sera.Ninety-eight percent (98%) of the E. coli isolates were agglutinatedusing the positive antisera in contrast to the negative sera. Thepositive antisera also reacted with Pseudomonas aeruginosa, Klebsiellapneumoniae and five sero groups of Salmonella (serotype B, C₁, C₂, D₁and E₃).

EXAMPLE 6 Serological Response to Siderophore Receptor Proteins (SRP) ofE. coli in Vaccinated and Non-Vaccinated Flocks Under Natural FieldConditions

Fifty one thousand, one-day old turkey poults were equally divided amongtwo barns designated as barns 1 and 2. At six weeks of age, birds inbarn 1 were subcutaneously injected with a water-in-oil vaccine asdescribed hereinabove in Example 2. Each bird received 0.5 cc containing300 μg E. coli serotype 078 siderophore receptor protein (SRP) in thelower neck region. Barn 2 remained as non-vaccinated controls. Blood wasdrawn from 15 birds per barn at weekly intervals.

FIG. 3 represents the serological response to E. coli SRPs betweenvaccinated and non-vaccinated flocks. The antibody response to the SRPsin the vaccinated flock increased steadily with each sampling period ascompared to non-vaccinated controls. At 35 days following vaccination,the vaccinated group had a 7.1 times greater antibody response than thecontrol group.

Table 1, below, shows the average weight of processed birds between thevaccinated and non-vaccinated flocks. There was a statistically greaterweight advantage between the vaccinated flock (12.2 lbs/bird) ascompared to the non-vaccinated flock (11.8 lbs/bird). TABLE I THEAVERAGE BODY WEIGHT BETWEEN SRP-VACCINATED AND NON-VACCINATED TURKEYS ATTIME OF PROCESSING Barn 2 (non-vaccinated) Barn 1 (SRP-vaccinated) Ave.Body weight Ave. Body weight # of Birds/lot (Lbs) # of Birds/lot (Lbs)2772 11.85 1986 12.00 3108 11.91 3168 12.11 3024 11.92 3072 12.04 316811.97 3060 12.25 3256 11.98 3072 12.36 3186 11.75 3072 12.57 3136 11.653024 12.31 2112 11.42 3024 12.16 Total 23762 Mean 11.8 Total 23460 Mean12.2 SD 0.192 SD 0.18 CV 1.63 CV 1.54

FIGS. 4 and 5 show the total percent mortality and culls in E. coliSRP-vaccinated sister flocks (i.e., originating from the same breederhens or hatchmates), and the non-SRP-vaccinated controls, from 3-13weeks of age. These results show the true field mortality aftervaccination, by excluding early poult mortality which could result inerroneous results. As can be seen, there was a significant reduction inboth mortality and birds culled in the SRP-vaccinated flocks. Theseresults demonstrate the usefulness of E. coli-derived siderophorereceptor proteins in a vaccine for controlling systemic infectionscaused by E. coli under natural field conditions.

EXAMPLE 7 Cross-Reactivity of SRPs of Salmonella senftenberg andPasteurella multocida

Forty-eight Nicholas turkey poults were raised in isolation from one dayof age. At three weeks of age, the birds were divided into two equalgroups designated as Group 1 and Group 2. Twelve birds in Group 1 werevaccinated subcutaneously with (0.5 cc) 300 μg purified SRP isolatedfrom Salmonella senftenberg. The vaccine was prepared as described inExample 2 above. The remaining twelve birds were used as non-vaccinatedcontrols. Birds in Group 2 were treated the same as in Group 1, except12 of the birds were vaccinated with 300 μg purified SRP isolated fromPasteurella multocida.

Blood was taken from all of the birds in both groups at 5 day intervals.Fifteen days after the first injection, vaccinated birds received asecond injection of the appropriate SRP. Each vaccinated bird received500 μg, (0.5 cc) SRP subcutaneously in a water-in-mineral adjuvant. Allnon-vaccinated birds remained as controls. Birds were bled at 5-dayintervals.

Fifteen days after the second injection, the vaccinated birds in Group 1were intravenously challenged with 100 μg S. Heidelberg SRP (FIG. 7).Blood was taken at 2-day intervals post challenge. There was a highantibody response to challenge at 2- and 4-days post challenge. Thisdata shows the cross-reactivity of S. heidelberg to S. senftenberg.These proteins, in turn, both cross-react with E. coli, as demonstratedby the ELISA using E. coli SRPs as the capture antigen according to theprotocol described hereinabove in Example 5.

Likewise, 15 days after the second injection, all birds in Group 2 werechallenged intramuscularly with 1.1×10⁶ CFU of P. multocida, ATCC strainP-1059. Mortality was recorded daily for 2 weeks post-challenge. FIG. 6and Table 2 below also shows the mortality between the vaccinated andnon-vaccinated birds following challenge. TABLE 2 Mortality ofVaccinated and Non-Vaccinated Turkeys Following Challenge withPasteurella multocida P-1059 Numbers of dead/total tested Non-vaccinatedVaccinated 11/12 (91.6%) 1/12 (8.3%)

Eleven (91.6%) of the non-vaccinated birds died within 14 days afterchallenge (see, FIG. 6). In contrast, only 1 (8.3%) of the birds in thevaccinated group died. These results demonstrate that siderophorereceptor proteins can be used as protective immunogens.

FIGS. 7 and 8 show the serological response of birds vaccinated withsiderophore receptor proteins isolated from S. senftenberg and P.multocida, respectively. The siderophore receptor proteins inducedprimary and secondary immune responses in both vaccinated groups at 10and 20 days post-vaccination as compared to non-vaccinated controlbirds. These antibody responses demonstrate the cross-reactive nature ofthese protein, which was confirmed in the ELISA assay using SRPsisolated from E. coli as capture antigens.

EXAMPLE 8 Cross-Reactivity of Siderophore Receptor Proteins as Evaluatedby ELISA

The cross-reactivity of E. coli siderophore receptor proteins fromExample 7 above was further examined using an Enzyme-LinkedImmunosorbent Assay (ELISA). The siderophore receptor proteins (SRPs)were purified from polyacrylamide gels using a model 422 electro-eluter(Bio-Rad Laboratories, Hercules, Calif.). The proteins were then used ascapture molecules in an indirect ELISA test.

The optimum working concentrations of SRP and conjugate was determinedby several chequerboard titrations using positive and negative controlserums from Example 6 above. A prediction curve was then established tocalculate SRP ELISA titers at a 1:200 dilution. All subsequent testswere performed at a single serum dilution (1:200) and SRP titers werecalculated from the average of duplicate test absorbance values.

The ELISA was performed by adding 100 μl of diluted SRP of E. coli in0.05 M (0.1 μg) carbonate buffer (pH 9.6) to each well of a 96-wellflat-bottom, easy wash microtiter plate (Corning, Corning, N.Y.). Afterovernight incubation at 4° C., excess SRP was removed and the plate waswashed. All subsequent washing steps were done three times inphosphate-buffered saline (pH 7.4) with 0.05% Tween 20. The plates wereblocked for one hour at 37° C. with 4% Fish Gelatin (Sigma) in PBS andthen washed.

Duplicate serum samples from Example 7 were tested in parallel atsingle-point dilutions using 100 μl/well and incubated for 40 minutes at37° C. Each plate contained positive and negative control sera obtainedfrom birds from Example 4 above. After washing, 100 μlperoxidase-labeled conjugate was added to each well. After incubationfor 40 minutes at 37° C., the plates were washed and 100 μl of ABTSperoxidase substrate in buffered H₂O₂ solution (Kirkegaard & PerryLaboratories Inc., Gaithersburg, Md.) was added to each well. Thesubstrate was allowed to react for 15 minutes at room temperature. Thereaction was terminated with 50 μl of 1% SDS and the absorbance readdirectly using a MR6SO microtiter plate reader (Dynatech Laboratories,Alexandria, Va.).

EXAMPLE 9 Fermentation Protocol for Production of Siderophore ReceptorProteins

The following protocol was used to culture E. coli 078 (ATCC #55652)resulting in expression of six (6) siderophore receptor proteins.

An E. coli master seed stock was prepared by growing the organism in2000 ml of sterile BHT broth containing 1-500 μg 2,2′-dipyridyl for 8hours at 37° C. The bacteria were harvested by centrifugation at10,000×g for 30 minutes. The culture is washed twice by centrifugationand resuspending the pellet in sterile PBS. The final pellet wasresuspended into 500 ml sterile BHI containing 20% sterile glycerol. Onemilliliter of culture was transferred to a 2-ml cryovial and stored at−85° C.

A cryovial (1 ml) of the E. coli master seed stock was used to inoculatea 100-ml culture flask containing tryptone (10 g/l), yeast extract (5g/l) dextrose (2 g/l), NaCl (10 g/l), and 2,2′-dipyridyl (15.0 μg/ml).The culture was incubated at 37° C. for 7 hours, at which time it wasinoculated into 2 liters of the above media and allowed to grow for anadditional 4 hours at 37° C. The 2-liter culture was used to inoculate a20-liter Virtis bench-top fermenter (Model 233353, Virtis, Gardiner,N.Y.) charged with 13 liters of the above-described media. The pH washeld constant between 6.9 and 7.2 by automatic titration with 30% NaOHand 10% HCl. The stirring speed was 250 rev/minute, and the culture wasaerated with 11 liters/minute at 34° C. Foaming was controlledautomatically by the addition of 0.4% silicone defoamer (Antifoam-B, J.T Baker, N.J.). The culture was allowed to grow continuously at theseconditions for 12 hours (O.D. 600 nm=7.10) at which time it was pumpedinto a 150-liter fermenter (W. B. Moore, Eastón Pa.) charged with 110liters of the above-described media containing 26.7 μg/ml dipyridyl and0.2% defoamer. The conditions in the fermenter were as follows: 450 rpm,50 slpm air, 10 psi backpressure, 34° C., and pH held at 6.9 with NaOH.

After 12 hours of fermentation, the bacteria were inactivated by theaddition of 0.15% formalin. The bacteria were harvested by continuousflow centrifugation (20,000×g at 4° C.) using two Beckman (Model J2-21M)centrifuges equipped with JCF-Z continuous flow rotors.

The pelletized bacteria were then washed to remove contaminating culturemedia proteins and further processed as described above in Example 1.The concentrated material was treated with 0.2% SDS and eluted asdescribed above in Example 1. The peak from the elution profilecontaining approximately 85% pure siderophore receptor proteins wasethanol precipitated to remove SDS, and resuspended in PBS.

The material was separated by SDS-PAGE as described above in Example 4with a 4% stacking gel on 12.5% acrylam-de gel. The SDS-PAGE pattern ofthe outer membrane protein extract showed expression of SRPs havingmolecular weights of 91-92 kDa, 89 kDa, 84 kDa, 78 kDa, 74 kDa and 72kDa.

EXAMPLE 10 Efficacy of Vaccine of SRPs from Escherichia Coli UnderNatural Field Conditions

The efficacy of vaccinating turkeys with E. coli siderophore receptorproteins (SRPs) under natural field conditions was shown as follows. Afarm complex with a history of disease was chosen for experimentaltrials. The facility was a three state operation, having two broodingbarns and eight finishing farms.

Data was collected for one year prior to vaccination to establish anaccurate profile on mortalities and bird performance (flocks 1-16 beforevaccination) Vaccinating with SRPs was evaluated for a period of 6months (flocks 17-24 after vaccination). A total of 24 flocks comprising1,160,864 birds was examined. Vaccination trials began in January andran through July, considered to be a critical time period for E. coliinfections and other natural field challenges.

Brooder barns 1 and 2 were divided in half and designated as A and B(barn-1) and C and D (barn-2). Approximately 50,000 randomized hens wereplaced in each barn so that each flock contained 25,000 birds All flockswere vaccinated by subcutaneous injection at 3 weeks of age with avaccine preparation containing SRPs (MW 91-92 kDa, 89 kDa, 84 kDa, 78kDa, 74 kDa and 72 kDa, SDS-PAGE on 12.5% acrylamide gel) isolated andpurified from E. coli 078 as described above in Example 1. Flocks A andC were vaccinated with a dosage level of 300 μg SRP and 109 TCID₅₀Newcastle Disease Virus (NDV) in a water-in-oil emulsion. Flocks B and Dwere the controls, and given a dosage level of 10⁹ TCID₅₀ NDV only.

At 4 weeks of age, the birds were moved into four second-stage barnswhile maintaining identity. At nine weeks of age, the birds were movedto four finishing barns, keeping identity on each 25,000 bird flock.Birds were marketed at 12- and 14-weeks of age and identity wasmaintained throughout processing.

Table 3 shows the cumulative farm history before and afterSRP-vaccination. Twenty-four flocks were evaluated, the 16 beforevaccination (1-16) and the 8 vaccinated flocks (17-24) includingcontrols. Flocks 1-16 were not SRP-vaccinated and included as a farmhistory to show the performance advantage to SRP-vaccinated flocks17-24.

Table 3 below, shows the age at which each flock was marketed, the headcount, total percent mortality, condem (i.e., condemnation atprocessing), and average bird weight/lot processed. TABLE 3 FlockHistory Before SRP-vaccination Age Head Mortality Condem Flocks (days)Count (%) (%) Ave. wt. 1 97 47818 8.37 1.13 13.88 2 94 45638 12.53 1.1713.80 3 95 51443 12.87 3.44 13.53 4 96 49999 4.20 1.23 13.86 5 92 497334.68 0.96 13.25 6 96 48303 7.36 1.25 13.49 7 101 48722 16.50 2.12 15.103 103 51456 12.26 1.41 15.60 9 98 50423 7.84 1.63 14.73 10 96 50880 7.041.59 13.81 11 95 46710 14.85 1.16 14.04 12 98 48994 11.32 1.09 13.89 1392 43433 21.28 1.74 13.23 14 94 49806 9.59 1.08 13.64 15 93 39216 28.082.35 12.92 16 96 46119 15.95 1.45 13.76

Flock History After SRP-vaccination Age Head Mortality Condem Flocks(Days) Count (%) (%) Ave. wt. 17 99 48323 8.08 1.45 15.37 18 96 480918.15 1.16 14.93 19 96 48743 6.89 1.07 16.16 20 90 48462 7.36 1.06 14.1121 92 49175 6.11 1.00 15.08 22 90 48261 7.86 0.83 14.38 23 94 51813 5.950.92 15.52 24 98 49296 9.44 1.08 16.10 Mean 96/94 48043/49021 12.2/7.5  1.5/1.07 13.9/15.3 SD 2.9/3.5 6.2/1.2 0.83/0.13 0.70/0.77 CV 3.1/3.751.4/15.5 40.9/17.2 5.0/5.0

As shown above in Table 3, the average percent mortality beforevaccination was 12.2±6.2 with a coefficient of variation (cv) of 51.4%as compared to the average mortality after vaccination of 7.5±1.2 with acv of 15.5%. This is a 4.7% decrease in mortality, which equates to 4700birds for every 100,000. The decrease in the coefficient of variation(51.4% as compared to 15.5%) on total mortality illustrates a positiveeffect on bird livability and uniformity. FIG. 9 is a graphicalrepresentation of mortalities in consecutive flocks before and aftervaccination.

Condemnation was also positively effected showing 1.6±0.63 percentbefore vaccination as compared to 1.07±0.18 percent after vaccination(Table 3 above). The difference, 0.53% is significant considering thenumber of birds processed.

A dramatic effect that was observed by the SRP vaccination was theincreased weight advantage, as seen above in Table 3. Before vaccinationthe average bird weight was 13.9±0.70 pounds, with an average growingtime of 96 days. The average weight per bird after vaccination was15.3±0.77 pounds, with an average growing time of 94 days. These resultsdemonstrate the advantage in performance that can be obtained throughSRP-vaccination.

FIG. 10 shows the serological response to SRPs of E. coli between theSRP-vaccinated and non-SRP-vaccinated flocks as determined by ELISA,using purified E. coli SRPs as the capture molecule. The assay wasconducted as described above in Example 8. The profile was consistentbetween the vaccinated and non-vaccinated flocks under natural fieldconditions. As the profile illustrates, once the bird's immune systembecomes focused to recognize these proteins, continuous field challengeby bacteria expressing SRPs causes a steady rise in antibody titer to alevel which provides protection and/or to the point where systemicchallenge does not effect performance.

Using purified IROMPs in a vaccine optimizes the animal's immune systemto focus on those proteins. The birds vaccinated with 300 μg purifiedSRP at three weeks of age showed an increase in titer at 11 weeks of agewhich was 10,000 times greater than the titer in the non-SRP-vaccinatedcontrols. This increase in titer is the result of focusing the immunesystem to recognize these proteins. Once vaccinated, the birdestablishes a population of memory cells that are activated upon eachfield challenge. Under natural field conditions, the bird iscontinuously challenged by gram-negative bacteria such as E. coli, whichexpress SRPs that cross-react and cause a continuous rise in antibodytiter (as was seen in the SRP-vaccinated birds). By comparison, thecontrol birds under the same conditions, show low antibody titers eventhough exposed to the same field challenges.

EXAMPLE 11 Vaccination with SRP-Vaccine and Vaccine Made with BacterialWhole Cells

A comparison was made between turkeys injected with a vaccine made ofpurified SRPs derived from Salmonella Heidelberg prepared as describedabove in Example 1, and a vaccine made of bacterial whole cells of thesame organism grown under iron-restrictions so as to express SRP on thecell surface. The whole cell bacteria was prepared as described inExample 1, except for the following modification: after the fermentationprocess 0.3% formalin were added to the vessel to kill the organism. Thekilled bacteria were collected as described in Example 1, washed andresuspended in physiological saline, and adjusted to an optical densityof 35% T at 540 mm to give approximately 10⁷ bacteria/ml The vaccine wasprepared as described above in Example 2.

Forty-five thousand one-day old hybrid turkey poults (hens) were raisedto 4 weeks of age, on a brooding facility. At four weeks of age, thebirds were moved to a growing facility and equally divided among twobarns designated as barns 1 and 2. At 6 weeks of age, birds in barn 1were vaccinated subcutaneously in the lower neck with 0.5 cc of the SRPvaccine while the birds in barn 2 were vaccinated with the whole cellpreparation. Blood was taken from 12 birds/barn at weekly intervals tomonitor the serological response to SRP between the two groups.

FIG. 11 shows the titer to SRP between whole cell and SRP-vaccinatedbirds. The immunological response to SRP was significantly greater inpurified SRP-vaccinated group as compared to the whole cell vaccinatedgroup. These results clearly demonstrate the efficacy of using asubstantially pure preparation of SRP for inducing an immune response inan animal in contrast to using whole cell expressing the same SRP.

EXAMPLE 12 Transfer of Anti-SRP Antibodies to Breeder Hen Progeny

The 10-day mortality in progeny from SRP-vaccinated and non-vaccinatedbreeder hens was evaluated to assess the transfer of anti-SRP antibodiesfrom adult to progeny.

Twenty thousand randomized Nicholas turkey poults (hens) were equallydivided among two brooder barns designated as barns 1 and 2. At fourweeks of age, all birds in barn 1 were vaccinated with 300 μg of E. coliSRP and Newcastle Disease Virus (NDV) in a water-in-oil vaccine. Birdsin barn 2 were given NDV only and acted as controls. At 24 weeks of age,the birds from barn 1 were given a second injection of SRP at 300μg/bird. Birds from barn 2 remained as non-vaccinated controls. Atthirty weeks of age, the birds were placed in barns 1 and 2 of a layingfarm. At mid-lay, eggs were collected from the SRP-vaccinated andnon-vaccinated hens. Eggs were set in separate incubators and hatchers.At hatch time, all poults were treated the same and identity wasmaintained throughout sexing and servicing.

Five thousand poults (hens) from each group were placed in a commercialbrooding barn and kept in brooding rings at 7 rings/group containing 714poults/ring. Poult mortality was monitored for each ring/group for aperiod of 10 days.

The total 10-day mortality in poults originating from the SRP-vaccinatedhens was 105 (2.1%) as compared to 160 (3.2%) in the non-vaccinatedprogeny (FIG. 12). This is a 1.1% advantage in poult livability, whichequates to 1100 poults for every 100,000. This is significantconsidering that there are 200 million turkeys in the United States and7 billion broilers worldwide.

These results show the beneficial effect of vaccinating breeding stockto induce maternal antibody to SRP in progeny to reduce gram-negativeinfections that are responsible for much of the early poult mortality.

EXAMPLE 13 Cross-Reactive and Cross-Protective Nature of SiderophoreReceptor Proteins (SRP) Between Different Serogroups of Salmonella

The SRP of Salmonella enteritidis (Se), serogroup D₁ and Salmonellatyphimurium (St), serogroup B were examined for their ability tocross-react and cross-protect. Briefly, 160 randomized hybrid turkeypoults (hens) were raised in isolation. At three weeks of age, the birdswere equally divided among 4 isolation rooms, 40 birds/room, designatedas A, B, C and D. Birds in group C were subcutaneously injected with awater-in-oil vaccine, as described hereinabove in Example 2, containing300 μg SRP of S. typhimurium. Birds in room D were subcutaneouslyinjected with 300 μg SRP of S. enteritidis. Birds in rooms A and Bremained as non-vaccinated controls. Blood was taken from 10 birds/groupat weekly intervals to monitor the serological response to SRP.

Twenty one days after the first injection, birds in groups C and D weregiven a second injection containing 300 μg of the appropriate SRP. Bloodwas taken at 5 and 10 days after the second injection. The serologicalresponse to SRP was examined by ELISA using E. coli SRP as the capturemolecule as described above in Example 8.

FIGS. 13 and 14 show the serological response of birds vaccinated withSRP isolated from S. typhimurium and S. enteritidis. The immunologicalresponse to SRP increased steadily in both groups with each samplingperiod as compared to the non-vaccinated controls, showing theimmunogenicity of these proteins. Importantly, these results show thecross-reactive nature of these proteins since the ELISA is using E. coliSRP as the capture molecule.

Fifteen days after the second injection, all birds were intravenouslychallenged with a nalidixic acid resistant strain of S. enteritidis orS. typhimurium at 5.0×10⁷ colony forming units (CFU)/bird. Thesebacteria were made resistant to nalidixic acid to enhance theirisolation by incorporating nalidixic acid in the recovery media whicheliminated any contamination. Bacteria resistant to nalidixic acid wereprepared as follows: One ml of a 12-hour Tryptic soy broth (TSB) cultureof S. enteritidis and/or S. typhimurium containing approximately 10⁸viable organisms, was spread over the surface of a brilliant sulfurgreen (BSG) agar (Difco) plate containing 500 μg/ml nalidixic acid(Sigma). The plates were incubated at 37° C. for 24 hours and thecolonies that grew were cloned by plating on BSG containing 250 μg/mlnalidixic acid. The nalidixic acid-resistant strains of salmonella wereincubated in 100 ml of TSB at 37° C. for 12 hours. At the end ofincubation, the culture was centrifuged (10,000×g) and washed twice inPBS (pH 7.4), and the optical density was adjusted to 35% transmissionat 540 nm to obtain 5.0×10⁷ CFU/ml. These isolates were then used forchallenge.

To evaluate homologous and heterologous protection, twenty birds in roomC (vaccinated with St-SRP) were wing banded and moved into room D, and20 birds in room D (vaccinated with Se-SRP) were wing banded and movedto room C. All birds in room C (20 St-vaccinated and 20 Se-vaccinated)were challenged with S. typhimurium, while birds in room D (20Se-vaccinated and 20 St-vaccinated) were challenged with S. enteritidis.

At 24, 48, 72 and 96 hours post-challenge, two birds from each groupwere killed. The spleens were aseptically removed from each bird andindividually weighed, and adjusted to 4 grams/spleen. A fecal samplefrom the cecal junction from each bird was also taken. Each sample wasweighed and adjusted to 0.5 grams. Four milliliters of sterile salinewas added to each spleen and 0.5 ml to each fecal sample. Each samplewas homogenized using a Stomacher Lab Blender (Sewert Medical, London)for 1 minute. Serial ten-fold dilutions of each homogenate were platedin duplicate on BSG plates containing 250 μg/ml nalidixic acin.

The results show the quantitative clearance of S. typhimurium (St) (FIG.15) and S. enteritidis (Se) (FIG. 16) in spleens of SRP-vaccinated andnon-vaccinated turkeys. As shown in FIGS. 15 and 16, there was a steadydecline in the number of bacteria/spleen. At 96 hours after challenge(chlg), the difference between the vaccinated and non-vaccinated groupswas approximately 2.5 logs. An important aspect of these results is thecross-protective nature induced by these proteins. FIG. 15 shows thecross-protective nature of the birds vaccinated with the SRP of Se butchallenged with St. FIG. 16 shows this same cross-protective effect ofbirds vaccinated with SRP of Se and then challenged with St. Allvaccinated groups showed a significant reduction in the number ofbacteria in spleens in contrast to the non-vaccinated birds.

At 72 and 96 hours after challenge, intestinal shedding of Salmonellawas detected in the non-vaccinated birds at greater then log 4. Incontrast, all of the vaccinated birds were negative for Salmonellawithin this same sampling period. These results indicate that theseproteins may have some beneficial effect in preventing the intestinalcolonization of Salmonella.

EXAMPLE 16 Preparation and Use of the 37-38 kDa Transmembrane and PorinProteins in a Vaccine

The transmembranes and porin proteins (MW 34-38 kDa), identified asOmpA, OmpC, OmpD and OmpF are expressed with and without iron. Theseproteins can be purified as described above in Example 1, by collectingfractions 1650-2250 as shown in FIG. 1. These proteins can be combinedwith peak 1 (FIG. 1) to obtain a combination of SRP and porin proteinsthat are conserved among Salmonella, E. coli, and Pasteurella.

A vaccine containing E. coli SRPs (MW 89 kDa, 84 kDa, 78 kDa and 72 kDa)was combined with porins (MW 34 kDa-38 kDa) to give a total proteincontent of 600 μg/ml, and prepared as described above in Example 2. Thevaccine was used to induce hyperimmunized sera. Briefly, six (6)three-week old turkeys were given a single subcutaneous injection in thelower neck region followed by a second injection 15 days after. Serumwas collected 10 days after the second injection.

Western blot analysis, as described above in Example 4, using sarcosinecell wall extracts of E. coli, Salmonella and Pasteurella and probedwith the above sera revealed cross-negative proteins in the 34 kDa and38 kDa region as well as the SRPs from each isolate examined

These results indicate the potential of using conserved protein (SRP andporins) as an effective method for vaccinating against gram-negativeinfections.

1-34. (canceled)
 35. A method for immunizing an animal, the methodcomprising administering to an animal a composition comprising:siderophore receptor proteins (SRPs) extracted from an outer membrane ofa bacterium family Enterobacteriaceae, family Pasteurellaceae, or familyPseudomonadaceae; wherein the SRPs have molecular weights of about 72-96kDa; a non-iron regulated protein obtained from an outer membrane of abacterium of family Enterobacteriaceae or family Pasteurellaceae,wherein the non-iron regulated protein has a molecular weight of about34-38 kDa; and a physiologically acceptable carrier.
 36. The method ofclaim 35 wherein the bacterium of family Enterobacteriaceae isEscherichia coli, and the composition comprises at least 2 SRPs.
 37. Themethod of claim 35 wherein the bacterium of family Enterobacteriaceae isSalmonella spp., and the composition comprises at least 3 SRPs.
 38. Themethod of claim 37 wherein the Salmonella spp. is serotype B, serotypeC₁, serotype C₂, serotype D₁, or serotype E₃.
 39. The method of claim 35wherein the bacterium of family Pasteurellaceae is Pasteurella spp. 40.The method of claim 35 wherein the Pasteurella spp. is Pasteurellamultocida, and the composition comprises at least 3 SRPs. 41-47.(canceled)
 48. A method for immunizing an animal, the method comprisingadministering to an animal a composition comprising: at least 3 SRPsextracted from an outer membrane of a first Salmonella spp., wherein theSRPs have molecular weights of about 72-96 kDa; a non-iron regulatedprotein obtained from an outer membrane of a bacterium of familyEnterobacteriaceae, wherein the non-iron regulated protein has amolecular weight of about 34-38 kDa; and a physiologically acceptablecarrier; wherein the composition cross-protects against a Salmonellaspp. infection in the animal caused by a second Salmonella spp. whereinthe first Salmonella spp. and the second Salmonella spp. are the samespecies.
 49. A method for immunizing an animal, the method comprisingadministering to an animal a composition comprising: at least 3 SRPsextracted from an outer membrane of a first Salmonella spp., wherein theSRPs have molecular weights of about 72-96 kDa; a non-iron regulatedprotein obtained from an outer membrane of a bacterium of familyEnterobacteriaceae, wherein the non-iron regulated protein has amolecular weight of about 34-38 kDa; and a physiologically acceptablecarrier; wherein the composition cross-protects against a Salmonellaspp. infection in the animal caused by a second Salmonella spp., whereinthe first Salmonella spp. and the second Salmonella spp. are differentspecies. 50-81. (canceled)
 82. The method of claim 48, the compositionfurther comprising at least one SRP extracted from the outer membrane ofa gram negative bacterium, wherein the gram negative bacterium isdifferent from the first Salmonella spp.
 83. The method of claim 49, thecomposition further comprising at least one SRP extracted from the outermembrane of a gram negative bacterium, wherein the gram negativebacterium is different from the first Salmonella spp.